Metalloproteinase activity at the cell surface can strongly influence cell sensitivity to extrinsic death vs. survival signals in a variety of cell types, yet the convergence of metalloproteinases and receptor signaling in the nervous system remains largely unexplored. TIMP-3 (tissue inhibitor of metalloproteinase-3) is a unique natural metalloproteinase inhibitor, in that it is a potent inhibitor of all known metalloproteinase sheddases, and has been shown to play a role in the regulation of receptor-mediated cell death in various non-neuronal cell types. TIMP-3 plays a pro-apoptotic role in many cell types through its ability to inhibit sheddases that target death receptors and their ligands. Our interest in the role of metalloproteinases in CNS damage following stroke led us to investigate the expression of TIMP-3 and the metalloproteinase sheddase, MMP-3, in a rodent model of focal cerebral ischemia. We found that while TIMP-3 and MMP-3 are expressed at low to non-detectable levels in the adult brain, their expression becomes markedly upregulated following ischemia, particularly in cortical neurons undergoing delayed apoptotic death. Using a tissue culture approach, we found that TIMP-3 and MMP-3 are constitutively expressed by embryonic cortical neurons in culture and modulate neuronal sensitivity to receptor-mediated apoptosis induced by the chemotherapeutic drug, doxorubicin (Dox). Metalloproteinase inhibition by TIMP-3 was found to be necessary for Dox-induced apoptosis, whereas addition of exogenous active MMP-3 markedly attenuated apoptosis and blunted death receptor-ligand interactions at the cell surface. These observations strongly implicate a physiologic role for TIMP-3 and MMP-3 in the regulation of receptor-mediated death in the nervous system. While metalloproteinase activity has previously been implicated in vascular damage following cerebral ischemia, the ability of metalloproteinases and their inhibitors to influence neuronal vulnerability to ischemic stress has not been studied. In the proposed studies, experiments are designed to establish a role for metalloproteinase activity in the regulation of receptor-mediated neuronal death following cerebral ischemia, and to explore underlying mechanisms, utilizing both in vitro and in vivo models of ischemic injury, coupled with pharmacological and gene deletion approaches.